Please choose Bioinformatics tool of your
interest from the internet. This tool can be either web based format that
you can submit your data in dialog box and get the result in real time,
or you may have to download and install the software on your hard disk.
Please show us each step, starting from submission your data until you
get the out put. You may press 'print screen' to copy, step by step and
paste those images on html file with the description. We will have your
result posted on HyperCourse homepage as soon as we get the URL from you.
No duplication of the utility or software will be allowed. Those who submit
the same software that already post on HyperCourse homepage may need to
find the new one.
The program used in these calculations was developed by Gerhard Steger
from Institut
fur Physikalische Biologie , for comparing theroretical predictions
to experimental data, mainly optical denaturation profiles, taken at 260
and 280 nm, and TGGE (temperature gradient gel electrophoresis) experiments.
The original version was written in VAX Fortran (VMS), using the Graphics
Kernel System GKS for data presentation (nice interactive program that
runs also with OpenVMS; available on request). A strict F77 text-only
version was produced from the full version, for use on 'the PC' (i8088,
no graphics; the minimal PC ...), but also useful on other small systems.
The HUSAR
staff at the DKFZ (German Cancer Research Center in Heidelberg) did a
first port to the GCG environment on their Convex system, which they passed
back to us for further improvement. This GCG version, now running on a
DEC AXP system (OpenVMS) and GCG V7.1, is available on request.
The WWW server version is based on the F77 version, compiled by GNU f77
on a Linux station; user interface and graphics output is produced by
Tcl/Tk. The basic PostScript output is converted to GIF by ImageMagick.
Program-specific informations:
Calculation is based on D. Poland's algorithm including the modification
by Fixman & Freire in the implementation described by Gerhard Steger.
The Poland algorithm calculates the denaturation profile for double-stranded
nucleic acid using nearest-neighbor stacking interactions and loop entropy
functions described in the literature.
The input data required for calculation are:the sequence, of course
(and no default here!), optional mismatched positions, the strand concentration,
affecting the dissociation temperature, the method to calculate the
final dissociation into single strands,the thermodynamic parameter set
(DNA/DNA low salt/RNA), and the temperature range in which the calculation
is performed.
In case you need access to the full range of input options, more options
are available to the experts.
Data sets predicted and figures drawn by the program are described below;
see also for OUTPUT.
A perspective view on the temperature-dependent denaturation profile
(denaturation probability vs. sequence position vs. temperature. This
plot does not include the dissociation of dsNA into single-strands;
thus it shows most clearly the relative stability of the different parts
of the NA.
The temperature-dependent relative UV hypochromicities as measured in
optical melting, at wavelengths of 260 and 280 nm (282 nm in case of
DNA), respectively (full hypochromicity corresponds to approx. 30% of
the OD at low temperature).
The derivative form of above hypochromicities, showing the melting temperature(s)
and corresponding half witdh(s) of the transitions(s), giving hints
about the transition cooperativity.
Predicted relative gel mobility, calculated according to Lerman et al.,
vs. temperature for different values of the 'retardation length' parameter
Lr. This plot can be used for direct comparison with TGGE experiments;
superpositions of plots generated with or without mismatched positions
given are useful as a hint whether specific mismatched duplexes could
be detected among homoduplexed DNA in a mixture of sample and reference
double strands having undergone a denaturation-renaturation cycle, using
either perpedicular or parallel TGGE.
A 'half-denaturation temperature' plot showing the temperature at which
each base pair has a probability of 50% to be in the open state. Similar
to the three-dimensional plot, this plot can be used to estimate the
destabilizing effect of mismatches on the surrounding part of the sequence:
a temperature-shift of the TGGE transition can be expected if the lowest
melting part of the sequence is directly affected by the mismatch.
Calculations can be done for oligonucleotides (>15 bases) or long
double strands (>50 bases), respectively. In the case of oligonucleotide
mode, a length-dependent correction for the strand dissociation process
is applied. We do not have sufficient experimental results to stringently
check for this mode to give valid results, but for the length range
of about 20 nucleotides there is at least experimental evidence. Using
'oligo' mode with far longer sequences gives misleading results!
References for Poland Service:
- Description of implemented programs
(You should cite this reference in your publication!):
Steger, G. (1994). Nucleic Acids Res. 22, 2760-2768.
Thermal denaturation of double-stranded nucleic acids: prediction of temperatures critical for gradient gel electrophoresis and polymerase chain reaction.
Abstract:
A program is described which calculates the thermal stability and the denaturation behaviour of double-stranded DNAs and RNAs up to a length of 1000 base pairs. The algorithm is based on recursive generation of conditional and a priori probabilities for base stacking. Output of the program may be compared directly to experimental results; thus the program may be used to optimize the nucleic acid fragments, the primers and the experimental conditions prior to experiments like polymerase chain reactions, temperature-gradient gel electrophoresis, denaturing-gradient gel electrophoresis and hybridizations. The program is available in three versions; the first version runs interactively on VAXstations producing graphics output directly, the second is implemented as part of the HUSAR package at GENIUSnet, the third runs on any computer producing text output which serves as input to available graphics programs.
- Original version of algorithm:
Poland, D. (1974). Biopolymers 13, 1859-1871.
Recursion relation generation of probability profiles for specific-sequence macromolecules with long-range correlations.
Abstract:
The problem of calculating detailed probability profiles giving the probability of each unit in the chain to be in the ordered state (and all other average quantities as well including the fraction of strand association) for specific-sequence macromolecules requiring statistical weights that correlate up to the total number of units in the chain (e.g., DNA, collagen) is formulated in terms of recursion relations for appropriate a priori and conditional probabilities, thus generalizing the approach of Lacombe and Simha for nearest-neighbor correlations in specific sequence macromolecules. The technique allows the probability profiles for chains of thousands of units to be calculated in minutes making no approximations in the basic model.
